The present invention relates generally to the field of agriculture.
Hemoglobins are widespread throughout the biosphere. (See Wittenberg and Wittenberg, 1990, Ann. Rev Biophys Chem. 19:217-241). They are found in a broad range of organisms from bacteria, through unicellular eukaryotes, to plants and animals, suggesting that they predate divergence of life into plant and animal forms.
Plant hemoglobins have been classified into symbiotic and nonsymbiotic types (Appleby, 1992, Sci Progress 76:65-398). Symbiotic hemoglobins are found in plants that are capable of participating in microbial symbioses, where they function in regulating oxygen supply to nitrogen fixing bacteria. Nonsymbiotic hemoglobins were discovered recently discovered and are thought to be the evolutionary predecessors of the more specialized symbiotic leghemoglobins. The ubiquitous nature of nonsymbiotic hemoglobins is evidenced by their broad presence across the plant kingdom. (See Appleby, 1985, Nitrogen Fixation and CO2 Metabolism, eds. Ludden and Burris, pp. 41-51).
The widespread presence and long evolutionary history of plant hemoglobins suggest a major role for them in the life of plants. Nonsymbiotic plant hemoglobins (nsHb), consisting of class 1, class 2, and truncated Hbs (class 3) are believed to be expressed universally in members of the plant kingdom. (Andersson et al, 1996, Proc Natl Acad Sci 93: 5682-5687; Watts et al, 2001, PNAS 98: 10119-10124).
The existence of plant hemoglobins in the root nodules of legumes for almost has been known for almost 60 years. (See, e.g., Kubo, 1939, Acta Phitochem 11:195-200; Keilen and Wang, 1945, Nature 155:227-229). Over the years, hemoglobins have been positively identified in three non-leguminous dicotyledonous plants: Parasponia andersonii, Tream tomentosa, and Casuarina glauce. (See, e.g., Appleby et al., 1983, Science 220:951-954; Bogusz et al., 1988, Nature 331:178-180; Kortt et al., 1988, FEBS Lett 180:55-60). Recently, an Hb cDNA from barley was isolated and the gene was demonstrated to be expressed in seed and root tissues under anaerobic conditions. (See Taylor et al., 1984, Plant Mol Biol 24:853-882). These observations support the viewpoint that plant hemoglobins have a common origin. (See Landsmann et al., 1986, Nature 324:166-168). Since Hb has been demonstrated to occur in two of the major divisions of the plant kingdom, it is likely that an Hb gene is present in the genome of all higher plants. (See Brown et al., 1984, J Mol Evol 21:19-32; Bogusz et al., 1988; Appleby, 1992, Sci Progress 76:365-398; Taylor et al., 1994, Plant Mol Biol 24: 853-862; Andersson et al., 1996, Proc Natl Acad Sci USA 93:427431; Hardison, 1996, Proc Natl Acad Sci USA 93:5675-5682).
The reported lack of effect of hemoglobin on cell growth and oxygen uptake under normal air conditions likely reflects the fact that barley (See Taylor et al., 1994, Plant Mol Biol 24: 853-862) and maize hemoglobin genes are induced under conditions of limited oxygen availability, resulting in the protein having little effect when oxygen supplies are not impaired. It has been shown clearly that the energy status of maize cells when oxygen is limiting is affected by the ability of the cells to produce hemoglobin. Total adenylates and ATP levels are maintained during the period of exposure to limiting oxygen when hemoglobin is constitutively expressed in the cells. (See WO 00/00597). Alternatively, when hemoglobin expression was suppressed by constitutive expression of antisense barley hemoglobin message, the cells were unable to maintain their energy status during oxygen limitation.
Class 1 nonsymbiotic hemoglobins are present in seed, root and stem tissue of monocots and dicots where they are expressed in response to hypoxia, etiolation, sucrose/mannitol addition, cytokinin, ARR1 or auxin (IAA) treatments in addition to nutrient oversupply (NO3−, NO2− and NO) and deprivation (P, K, and Fe). (See Taylor et al., 1994, Plant Mol Biol 24: 853-862; Hunt et al, 2001, Plant Mol Biol 47: 677-692; Lira-Ruan et al, 2001, Plant Sci 161: 279-287; Kim et al., 2003, Journal of Plant Biology 46: 161-166; Ohwaki et al., 2003, Plant and Cell Physiology 44: S78; Ross et al, 2004, J Exp Bot 55: 1721-1731; Wang et al, 2003, Plant Cell Environ 26: 673-680; Dordas et al, 2003, Plant Journal 35: 763-770). Class 1 nsHbs are also known to be repressed in roots following infection by mycorrhizal fungi. (See Uchiumi et al, 2002, Plant Cell Physiol 43: 1351-1358).
Hunt et al., 2002, PNAS 99: 17197-202, reported that A. thaliana over-expressing a class 1 A. thaliana nsHb (GLB1-high affinity) showed improved survival following severe hypoxic stress, and that similar A. thaliana plants transformed to over-express Parasponia class 1 Hb (GLB1S-medium affinity) demonstrated an intermediate level of hypoxic protection relative to controls and to plants transformed with GLB1 mutated to have a low affinity for gaseous ligands (GLB1 (HE7L)-low affinity).
More recent work with transgenic maize cell suspensions (Dordas et al, 2004, Planta 219: 66-72), alfalfa root cultures (Dordas et al, 2003, Plant Journal 35: 763-770; Igamberdiev et al, 2004, Planta 219: 95-102) and A. thaliana plants (Perazzolli et al, 2004, Plant Cell 16: 2785-2794) has demonstrated that class 1 nsHbs modulate plant NO levels, both in vitro and in vivo, with NO levels being inversely related to class 1 nsHb expression. Both barley and alfalfa class 1 nsHbs, together with a corresponding reductase, have been shown to metabolize NO to NO3−, with such activity being NAD(P)H-dependent and displaying characteristics of a NO-dioxygenase. (See Igamberdiev et al., supra; Seregelyes et al, 2004, FEBS Lett 571: 61-66).
Transgenic tobacco (Nicotiana tabacum) plants expressing a hypoxia-inducible bacterial hemoglobin (VHb) from the obligate aerobic, gram-negative bacteria Vitreoscilla have been shown to exhibit reduced emergence time, enhanced growth, accelerated development and increased chlorophyll content relative to control plants (Holmberg et al. 1997, Nature Biotechnol 15: 244-247). Petunias (Petunia hybrida) and tobacco plants expressing VHb also have demonstrated improved hydroponic growth and waterlogging tolerance relative to control plants. (See Mao et al. 2003, Acta Botanica Sinica 45: 205-210). VHb is a bacterial hemoglobin, and is separate and distinct from the plant nonsymbiotic hemoglobins encompassed by the present invention. For example, VHb has different biochemical properties than nsHb, has different ligand-binding properties, and has a lower oxygen affinity.
Hunt et al., 2002, supra, reported that that GLB1-transformed plants exhibited increased early growth (i.e., at 14 days), greater root and shoot weight at 14 days, and had longer roots with a lower root hair density and more lateral roots than control plants, when the transformed plants were grown under normal oxygen conditions. However, no altered development rate of leaf production was observed. Additionally, Hunt et al. found no differences in morphological development of A. thaliana plants expressing either Arabidopsis or Parasponia class 1 nsHb. The authors hypothesized that, although the plant was grown under normoxic conditions, the plant may have experienced localized, transient hypoxia, noting that a transient hypoxic phase may be experienced during germination. They therefore associated the observed effects on early root growth as being due to the transformed plant's improved ability to withstand that hypoxia.
While the effects of nonsymbiotic hemoglobin on oxygen uptake, NO levels, and survival under hypoxic conditions have been studied, the ability to modify plant phenotypes or mineral nutrition under normal oxygen conditions by controlling levels of nonsymbiotic hemoglobin has not heretofore been determined. Indeed, conflicting reports exist as to the influence of class 1 nsHb and/or VHb expression on plant growth under non-stressed, as compared to stressed, conditions. (See, e.g., Seregelyes et al. 2004, Febs Letters 571: 61-66; Haggman et al. 2003, Plant Biotechnology Journal 1: 287-300; Frey et al. 2004; Perazzolli et al. 2004, Plant Cell 16: 2785-2794).
There are a number of different plant phenotypes that it would be useful to be able to modify. For example, apical dominance in shoots and roots, taproot width, leaf size, leaf length, petiole length, internode length, plant shape, erect versus prostrate growing habit, flower color, early versus late flowering, chlorophyll content, and nutrient uptake, concentration, or metabolism.